We have investigated the pathogenetic mechanism of the mitochondrial tRNA^Lys gene mutation (position 8344) associated with MERRF encephalomyopathy in several mitochondrial DMA (mtDNA)–less cell transformants carrying the mutation and in control cells. A decrease of 50–60% in the specific tRNA^Lys aminoacylation capacity per cell was found in mutant cells. Furthermore, several lines of evidence reveal that the severe protein synthesis impairment in MERRF mutation–carrying cells is due to premature termination of translation at each or near each lysine codon, with the deficiency of aminoacylated tRNA^Lys being the most likely cause of this phenomenon.